In vitro studies of retroviral transduction of human and nonhuman primate hematopoietic cells have generally evaluated transduction of erythroid and mylomonocytic progenitors as assessed following culture in methylcellulose. Efforts to analyze the ability of transduced progenitor cells to undergo T cell differentiation have been limited by the difficulty in supporting T cell differentiation in vitro. We have recently developed an in vitro culture system using thymic stromal cultures from fetal rhesus macaques that supports in vitro T cell differentiation of human and rhesus CD34+ hematopoietic cells. We utilized this system to evaluate the effect of retroviral culture conditions on the ability of CD34+ cells to undergo T cell differentiation. Exposure of CD34+ cells to supernatant from the PG13 retroviral packaging cell line or to the combination of the cytokines IL-3, IL-6 and stem cell factor resulted in a dramatic inhibition of the ability of these cells to subsequently undergo T lymphopoiesis. In contrast, retroviral transduction of CD34+ cells in the presence of irradiated bone marrow stroma maintained T cell differentiation capacity and resulted in T cells expressing the marker gene neo. These results demonstrate the conditions commonly used for retroviral transduction of hematopoietic cells result in a significant loss of T cell differentiation capacity and possibly primitive pluripotent cells.